CRL4泛素化酶在卵巢癌中的功能和突变研究
发布时间：2018-10-31 06:26
【摘要】：目的研究泛素化酶CRL4蛋白复合体家族成员CRL4-WD40重复序列结构域蛋白70(WDR70)在卵巢癌细胞中的DNA修复功能,以及卵巢癌组织中该泛素化酶基因的突变规律。方法利用免疫荧光方法,检测CRL4骨架蛋白DDB1及WDR70基因特异性沉默的卵巢癌细胞与其对应的对照组细胞在化疗药物或放射线照射诱导产生DNA双链断裂后,组蛋白H2AX(γH2AX)及单链DNA结合蛋白32(RPA32)磷酸化灶点显示的差异;BrdU标记和染色实验检测WDR70基因对DNA复制是否存在影响,同时利用免疫组化染色检测卵巢癌组织临床病理标本及正常卵巢组织标本中的WDR70和组蛋白H2B单泛素化(uH2B)染色差异,以阐明CRL4的DNA损伤应答特征,RT-PCR测定卵巢癌组织中WDR70的基因表达水平,并采用DNA测序确定WDR70突变位点。结果免疫荧光染色结果显示,CRL4-WDR70的不同蛋白亚基(DDB1、WDR70)在细胞周期检验点激活和uH2B介导的DNA末端回切过程中起着不同的作用:DDB1参与以上两个机制的调控,而WDR70只促进末端回切、RPA32在DNA断裂点的招募和同源重组修复。BrdU标记和染色结果显示WDR70基因对DNA复制并不存在影响。免疫组化结果显示,卵巢癌组织临床病理标本及正常卵巢组织标本中的WDR70和uH2B表达存在差异。RT-PCR结果显示WDR70基因的全长、5′和3′转录本水平在50%的卵巢癌组织中水平减低,出现多处外显子突变位点。结论 CRL4在DNA修复过程中具有促进H2B单泛素化、促进DNA末端回切和激活细胞周期检验点等多种重要功能,是维持基因组稳定性、遏阻卵巢癌发生的重要抗癌机制。
[Abstract]:Objective to study the DNA repair function of CRL4-WD40 repeat domain protein 70 (WDR70), a member of the Ubiquitin CRL4 protein complex family, in ovarian cancer cells and the mutation rule of the ubiquitin gene in ovarian cancer cells. Methods CRL4 skeleton protein DDB1 and WDR70 gene specific silencing ovarian cancer cells and their corresponding control cells were detected by immunofluorescence method after DNA double strand breaks were induced by chemotherapeutic drugs or irradiation. The difference of phosphorylation sites between histone H2AX (纬 H2AX) and single-stranded DNA binding protein 32 (RPA32) was observed. The effect of WDR70 gene on DNA replication was detected by BrdU labeling and staining. Immunohistochemical staining was used to detect the difference between WDR70 and histone H2B monoubiquitin (uH2B) staining in clinicopathological specimens of ovarian cancer and normal ovarian tissues. In order to elucidate the characteristics of DNA damage response of CRL4, RT-PCR was used to detect the expression of WDR70 gene in ovarian cancer tissues, and DNA sequencing was used to determine the WDR70 mutation site. Results the results of immunofluorescence staining showed that different protein subunits (DDB1,WDR70) of CRL4-WDR70 played different roles in the activation of cell cycle detection points and DNA terminal recutting mediated by uH2B. DDB1 was involved in the regulation of these two mechanisms. However, WDR70 only promoted terminal reverse cutting, RPA32 recruitment at DNA break point and homologous recombination repair. The results of BrdU labeling and staining showed that WDR70 gene had no effect on DNA replication. Immunohistochemical results showed that the expression of WDR70 and uH2B were different between clinicopathological specimens of ovarian cancer and normal ovarian tissues. RT-PCR results showed the full length of WDR70 gene. The levels of 5 'and 3' transcripts were decreased in 50% of ovarian cancer tissues, and several exon mutation sites were found. Conclusion CRL4 has many important functions in the process of DNA repair, such as promoting monoubiquitization of H2B, promoting DNA terminal reversion and activating cell cycle test points. It is an important anticancer mechanism to maintain genomic stability and inhibit the occurrence of ovarian cancer.
【作者单位】： 四川大学华西第二医院妇产科;出生缺陷与相关妇儿疾病教育部重点实验室(四川大学);
【基金】：四川省科技厅应用基础计划项目(No.2017FZ0034) 四川省科技厅软科学计划项目(No.2017ZR0169)资助
【分类号】：R737.31
本文编号：2301220
[Abstract]:Objective to study the DNA repair function of CRL4-WD40 repeat domain protein 70 (WDR70), a member of the Ubiquitin CRL4 protein complex family, in ovarian cancer cells and the mutation rule of the ubiquitin gene in ovarian cancer cells. Methods CRL4 skeleton protein DDB1 and WDR70 gene specific silencing ovarian cancer cells and their corresponding control cells were detected by immunofluorescence method after DNA double strand breaks were induced by chemotherapeutic drugs or irradiation. The difference of phosphorylation sites between histone H2AX (纬 H2AX) and single-stranded DNA binding protein 32 (RPA32) was observed. The effect of WDR70 gene on DNA replication was detected by BrdU labeling and staining. Immunohistochemical staining was used to detect the difference between WDR70 and histone H2B monoubiquitin (uH2B) staining in clinicopathological specimens of ovarian cancer and normal ovarian tissues. In order to elucidate the characteristics of DNA damage response of CRL4, RT-PCR was used to detect the expression of WDR70 gene in ovarian cancer tissues, and DNA sequencing was used to determine the WDR70 mutation site. Results the results of immunofluorescence staining showed that different protein subunits (DDB1,WDR70) of CRL4-WDR70 played different roles in the activation of cell cycle detection points and DNA terminal recutting mediated by uH2B. DDB1 was involved in the regulation of these two mechanisms. However, WDR70 only promoted terminal reverse cutting, RPA32 recruitment at DNA break point and homologous recombination repair. The results of BrdU labeling and staining showed that WDR70 gene had no effect on DNA replication. Immunohistochemical results showed that the expression of WDR70 and uH2B were different between clinicopathological specimens of ovarian cancer and normal ovarian tissues. RT-PCR results showed the full length of WDR70 gene. The levels of 5 'and 3' transcripts were decreased in 50% of ovarian cancer tissues, and several exon mutation sites were found. Conclusion CRL4 has many important functions in the process of DNA repair, such as promoting monoubiquitization of H2B, promoting DNA terminal reversion and activating cell cycle test points. It is an important anticancer mechanism to maintain genomic stability and inhibit the occurrence of ovarian cancer.
【作者单位】： 四川大学华西第二医院妇产科;出生缺陷与相关妇儿疾病教育部重点实验室(四川大学);
【基金】：四川省科技厅应用基础计划项目(No.2017FZ0034) 四川省科技厅软科学计划项目(No.2017ZR0169)资助
【分类号】：R737.31
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